6-(alpha-(ome ga-guanidinoalkanoylamido)acylamido)penicillanic acids

ABSTRACT

6-( Alpha -( omega GUANIDINOALKANOYLAMIDO)ACYLAMIDO)PENICILLANIC ACIDS, THE NONTOXIC SALTS AND ESTERS THEREOF ARE USEFUL AS ANTIBACTERIAL AGENTS, THERAPEUTIC AGENTS IN ANIMALS, INCLUDING MAN, OF PARTICULAR VALUE AGAINST GRAM-NEGATIVE BACTERIA, AND AS ANIMAL FEED NUTRITIONAL SUPPLEMENTS.

United States Patent Hamanaka [75] Inventor: Ernest S. Hamanaka, Groton, Conn.

[73] Assignee: Pfizer Inc., New York, NY.

[22] Filed: Feb. 22, 1972 [21] Appl. No.: 228,344

[52] US. Cl. 260/239.l, 424/271 [5 1] Int. Cl C07d 99/16 [58] Field of Search 260/239.1

[56] References Cited UNITED STATES PATENTS 3,579,501 5/l97l McGregor 260/239.l

[451 Mar. 11; 1975 Primary Examiner-Nicholas S. Rizzo Attorney, Agent, or FirmC0nnolly and Hutz [57] ABSTRACT 6-[a-(m-guanidinoalkanoylamido)acylamidolpenicillanic acids, the non-toxic salts and esters thereof are useful as antibacterial agents, therapeutic agents in animals, including man, of particular value against gram negative bacteria, and as animal feed nutritional supplements.

13 Claims, N0 Drawings 1 6-(ALPHA-(OME GA-GUANIDINOALK- ANOYLAMIDO)ACYLAMIDO)PENICILLANIC ACIDS BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates to novel antibacterial agents; namely, (substituted acyl)derivatives of a-aminoacyl penicillins. More particularly, it is directed to 6-[a-(wguanidinoalkanoylamido)acylamido]penicillanic acids, the non-toxic salts and esters thereof, which are especially useful in the treatment of gram-negative infections and, particularly, Pseudomonas infections.

2. Description of the Prior Art A large number of 6-(a-aminoacylamido)penicillanic acids wherein the acyl moiety is alkanoyl or substituted alkanoyl wherein the substituent is an aryl, cycloalkyl or heterocyclic group are disclosed in U.S. Pats. Nos. 2,985,648; 3,007,920; 3,192,198; 3,485,819; 3,342,677; 3,538,083; 3,553,201; British Pats. Nos. 873,049; 903,785; 991,586; 1,033,257 and 1,189,990. Further, 6-[oz-substituted amino)acylamido]penicillanic acids are described in U.S. Pats. Nos. 3,198,788; 3,248,387; 3,308,023; 3,320,240; 3,325,477; 3,340,252; 3,381,001; 3,433,784; 3,518,253; British Pats. Nos. 891,977; 894,457; 985,688; 1,048,907; 1,051,675; 1,057,697; 1,064,893; 1,066,107; 1,125,339; 1,180,745; 1,210,472; Belgian Pat. No. 593,295 and Japanese Pat. No. 7,116,994. Addition-v ally, 6-(a-ureido acylamido)penicillanic acids are disclosed in U. S. Pat. No. 3,352,851 and German Pat. No. 2,054,772; 6-(a-guanidinoacylamido)penicillanic acids in U.S. Pats. Nos. 3,454,557 and 3,406,185; and a variety of p-guanidinoaroyl-, p-guanidinomethylaroyland p-guanidinoarylalkanoylamidopenicillanic acids are disclosed in U.S. Pat. No. 3,453,265. British Pat. No. 1,061,335 discloses 6-(D-a-hydrazinocarbonylaminoa-phenylacetamido)penicillanic acid, and British Pat. No. 1,053,818 describes esters of 6-(aoxalamidoacylamido)penicillanic acids.

A wide variety of 6-[oz-(substitute ureido)acylamido]penicillanic acids and 6-[a-(3-sub stituted thioureido)acylamido]penicillanic acids are reported in the recent literature. U.S. Pats. Nos. 3,479,339; 3,481,922; Netherlands Pats. Nos. 69,01646; 69,08909; and Japanese Pat. No. 7,112,732 describe such compounds wherein the 3-substituent is a carbamoyl group; the compounds being referred to as 6-[a-(3- allophanamido)acylamidolpenicillanic acids. U.S. Pat No. 3,579,501 disclosed 6-[a-(3- guanylureido)acylamidolpenicillanic acids; that is, such compounds wherein the 3-substituent is a guanyl moiety.

The above described products are active as antibacterial agents against a variety of gram-positive and gram-negative bacteria. However. while they are active in vitro and in vivo via the intraperitoneal route of administration, they are inactive or, at best, poorly active in vivo via the oral route of administration. Additionally, their pharmaco-kinetics, as evidenced by levels of the compound in the blood, are poor.

Summary of the Invention There has now been found a novel series of antibacand the pharmaceutically acceptable acid addition salts thereof wherein R is selected from the group consisting of hydrogen and acyloxy lower alkyl wherein acyloxy is selected from the group consisting of lower alkanoyloxy, benzoyloxy and substituted benzoyloxy wherein the substituent is selected from the group consisting of chloro, bromo, fluoro, lower alkyl, lower alkoxy and trifluoromethyl;

R, is selected from the group consisting of hydrogen, alkyl of l to 14 carbon atoms, cycloalkyl of 3 to 6 carbon atoms, 1,4-cyclohexadicnyl, naphthyl, benzyl, phenethyl, indolylmcthyl, furyl, thienyl, w-cthylthio(- lower alkyl and wherein Y is selected from the group consisting of hydrogen, nitro, di(lower alky1)amino, lower alkanoylamino, lower alkyl, lower alkoxy, hydroxy, sulfamyl, chloro, bromo, fluoro, iodo and trifluoromethyl;

R is selected from the group consisting of hydroge and lower alkyl;

R and R when taken together with the carbon atom to which they are attached are cycloalkylidene of 3 to 10 carbon atoms;

each of R R R and R is selected from the group consisting of hydrogen, lower alkyl, benzyl and phenyl;

R and R when taken together with the guanyl moiety to which they are attached from a 5- or 6- membered heterocyclic ring selected from the group consisting of 2-imidazolyl, 2(2-imidazolinyl), 2- (1,4,5,6-tetrahydropyrimidinyl) and 2-pyrimidinyl;

X is selected from the group consisting of alkylene having from 1 to 7 carbon atoms, phenylene, cycloalkylene having from 3 to 9 carbon atoms, propenylene whose CH group is bound to the adjacent nitrogen, vinylene phenylene, methylene oxyphenylene and phenylenemethylene, each of whose phenylene group is bound to the adjacent nitrogen;

X and N-R when taken together form a 5- or 6- membered heterocyclic ring selected from the group consisting of pyrrolidyl and piperidyl;

X and R when taken together with the guanidino moiety to which they are attached form a 5- or 6- membered heterocyclic ring selected from the group consisting of 2-amino-2-imidazolinyl and 2-amino-2- (l,4,5,-tetrahydropyrimidinyl);

R and R when taken together with the guanidino moiety to which they are attached form a 5- or 6- membered heterocyclic ring selected from the group consisting of 2imidazolino and 2-( l,4,5,6- tetrahydropyrimidino); and

R and R when taken together with the guanidino moiety to which they are attached form a 5- or 6- membered heterocyclic ring selected from the group consisting of imidazolidino and hexahydropyrimidino.

Inculded among the pharmaceutically acceptable acid addition salts of this invention are inorganic and organic acid addition salts. Typical salts include hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, acetate, citrate, benzoate, maleate, succinate, malate, fumarate, ascorbate, glycolate, tartrate, oxalate and mandelate.

Also included within the scope of this invention are the optically-active isomeric forms and mixtures thereof which arise by virtue of the asymmetric oz-carbon atom of the acyl side chain. These are the D- and the L-diastereoiosmers and the DL-form which is a mixture of the two optically-active isomers. The D- form of these compounds is the preferred form because of its greater activity relative to that of the L- or the DL-forms.

Other isomers of the herein described compounds in addition to those arising from the asymmetric a-carbon of the acyl side chain are, of course, possible because of the presence of asymmetric carbon atoms in the 6- aminopenicillanic acid nucleus.

The novel antibacterial products of this invention are of value as additives to materials such as fuels and cutting oils which are subject to bacterial deterioration and are useful in soaps and shampoos and in topical compositions for treatment of wounds. They are also remarkably effective in treating a number of infections caused by susceptible gram-negative and gram-positive bacteria in poultry and animals, including man.

DETAILED DESCRIPTION OF THE INVENTION The novel and valuable compounds of this invention are prepared by reacting an appropriate 6-[(a-aminosubstituted)acylamido]penicillanic acid, or a suitable ester thereof, of the formula 2 ca 1 3 rz-c-co-na-cu-ca c II 1 I I I (:a

2 O=CN CH-COOR with a reactive functional derivative of the carboxy group of an appropriate guanidino substituted acid of the formula R SNR n In Z-C-X-N-C-NRR 5 6 III wherein the variables X, R and R R are as defined above, and Z is hydroxy or a halo group.

Alternatively, compounds of formula I, especially those wherein X is alkylcne, can be prepared by reacting a compound of formula Ill-A with an appropriate S-alkylisothiourea of formula III-B S-alkyl RsRoN-L -NR; III-B according to standard procedures.

The terms lower alkyl, lower alkoxy and lower alkanoyloxy as used herein are intended to include those alkyl, alkoxy and alkanoyloxy groups having from 1 to 4 carbon atoms.

Suitable esters of the formula II reactants are those wherein R is acyloxy lower alkyl as defined above and those wherein R is a group which can readily be removed as, for example, by catalytic hydrogenation (benzyl, cyanomethyl, phenacyl, allyl and diphenylmethyl).

Suitable reactive functional derivatives of acids of formula III are the acid chlorides or bromides (Z Cl, Br). The acid reactant can be reacted with a condensing agent such as a carbodiimide, an alkoxyacetylene, N,N'-carbonyldiimidazole, N,N-carbonylditriazole and hexahalocyclotriphosphatriazines to give a reactive intermediate which is coupled to the 6-[(a-amino substituted)acylamido]penicillanic acid. Additionally, the appropriate acid azide or an active ester or thio ester of the formula III reactant with, for example, N- hydroxyphthalimide, N-hydroxysuccinimide, a phenol or thiophenol can be used as acylating agent.

The preferred acylation processes of this invention comprise the reaction of the appropriate 6-[(a-amino substituted)acylamido]penicillanic acid compound (formula II) with the acid chloride of an acid of formula III, or with the acid form of a formula III reactant in the presence ofa carbodiimide for reasons of come nience, availability of reactants and overall yield of product.

The 6-[(a-amino substituted)acylamido]penicillanic acid reactant can be used in a variety of forms. It can, for example, be used as the free acid or as an alkali metal or amine salt thereof. The use of a salt form of the penicillanic acid reactant is frequently of advantage since the solubility can be manipulated by judicious choice of the salt to permit the use of aqueous or nonaqueous systems. Alkali metal salts are valuable for use in aqueous systems. In non-aqueous systems, an amine salt such as a tertiary lower alkylamine salt, e.g., triethylamine, or an N-alkyl piperidine salt is generally used. Alternatively, an ester of the 6-[(a-amino substituted- )acylamidolpenicillanic acid is used, especially in nonaqueous systems. In those instances wherein the final product (formula I) is desired in the form of an ester (R is other than hydrogen), it is obvious and practical to use that ester form of the penicillanic acid reactant.

The acylation process is conducted in a reactioninert solvent system which can be aqueous or nonaqueous. Aqueous or non-aqueous solvent systems can be used when a carbodiimide is the condensing agent. When using a carbodiimide in an agueous system, the pH is desirably adjusted to the range of about 5 to about 8, and preferably to about 6 to 7. In a typical procedure, the formula III reactant and carbodiimide are mixed in equimolar proportions in a suitable solvent (tetrahydrofuran, dioxan) and a watcr-water-misciblc organic solvent solution (water plus dioxan or tctrahydrofuran) containing the 6-[(a-amino substituted- )acylamido1penicillanic acid is added at room temperature and the mixture stirred for several hours until reaction is complets. Temperatures of from about 5 to 30 C. are generally used. In most instances, an excess up to about of the condensing agent is used. The penicillin product is recovered by methods known to the art.

Acylation with an acid halide (formula III) can also be conducted in aqueous or non-aqueous solvent systems. In aqueous systems, the reaction is generally carried out at a pH of from about 6 to about 9 and a temperature of from about 0 C. to about 50 C. It can also be performed in unstable emulsions of water and a water-immiscible organic solvent such as methyl isobutyl ketone and lower alkyl acetates over the pH range of from about 2 to about 4.

In addition to the above purely chemical techniques of acylation, a sonochemical technique; that is, the application of vibrations of ultrasonic frequency (35,000 to 90,000 cycles per second), as described in US. Pat. No. 3,079,314, issued Feb. 26, 1963, can also be used to achieve acylation of the 6-[(a-amino substituted )acylamido1penicillanic acid, especially acylation with an acid halide. Acylation under such conditions is rapid and permissive ofa wide range of reaction media, aqueous and non-aqueous alike, homogeneous and nonhomogeneous, including emulsified systems.

The esters of this invention, compounds of formula I wherein R is acyloxy(lower alkyl), can be prepared by reacting an alkali metal salt (sodium, ptassium, lithium) of a compound of formula I wherein R is hydrogen and NR R is NHNO with the appropriate acyloxy(lower alkyl) halide (chloride or bromide). The reaction is normally conducted in a reaction-inert solvent such as tetrahydrofuran, dimethylformamide, dimethylsufoxide or hexamethylphosphoramide. In practice, the halide is added, usually dropwise, to a solution or suspension of an alkali metal salt of the nitroguanidino compound. At least one equivalent of the halide reactant is added but, in certain cases, it may be advantageous to employ as much as a 50 percent excess. The reaction is carried out at temperatures of from 0 to 50 C. and preferably from to 30 C. Reaction ktime will vary according to the temperature employed and the reactivity of the appropriate starting materials. Normally, the reaction period will range from 1 to 20 hours. The nitroguanidino ester derivative thus produced is then catalytically hydrogenated to the corresponding guanidino ester of formula I. The nitroguanidino derivative ofI (R=H, NR R NHNO is prepared by acylating the appropriate a-aminoacylpenicillin with the appropriate nitroguanidino carboxylic acid by methods described herein.

Alternatively, and preferably, the acyloxy(lower alkyl) esters of formula I compounds are prepared by the above described acylation procedures but using the appropriate acyloxy(lower alkyl) ester of the appropriate 6-[(a-amino substituted)acylamido]penicillanic acid in place of the non-esterified 6-[(a-amino substituted- )acylamido]penicillanic acid. The acyloxy(lower alkyl) esters of the 6 -[(a-amino substituted)acylamido]- penicillanic acids are prepared according to methods described in Belgian Pat. No. 721,515 and by Daehne et al., J. Med. Chem. 13, 607-612 (1970).

The acyloxy(lower alkyl) halides are synthesized from the corresponding acid chlorides and aldehydes or ketones in accordance with the general procedures of Ulrich et al., J. Am. Chem. Soc. 43. 660 (1921) and Euranto et al., Acta. Chem. Scand. 20, 1273 (1966). The formation of esters from acid salts and alkyl halides is well documented in the chemical literature (Zook and Wagner, Synthetic Organic Chemistry, John Wiley and Sons, Inc.,'New York, 1956, p. 484). The 6-[(a-amino substituted)acylamidolpenicillanic acid reactants are described in the art cited above. 5 Many of the guanidino substituted carboxylic acid reactants of the formula HOOCX-NR C(=NR- )NI-IR R are described in the literature. Particular reference is made to US. Pats. Nos. 3,257,41 1; 3,406,185 and 3,479,401 which describe suchacids wherein X is phenylene, methylenephenylene, alkylene and cyclohexylene (see formula II). Those guanidino substituted acid reactants which are not known in the art are prepared from the corresponding amino substituted carboxylic acids by reacting the amino acid with S-methylisothionitrourea. The intermediate nitroguanidinocarboxylic acid is then catalytically hydrogenated to the desired guanidino substituted carboxylic acid.

An alternative and favored method for preparing guanidino-substituted carboxylic acids comprises reaction of the appropriate amino substituted carboxylic acid with benzoylcyanamid followed by hydrolysis of the benzoyl guanidino substituted reaction product.

A further method for preparing the guanidinosubstituted carboxlic acids involves reaction of the appropriate amino substituted carboxylic acid with O- methylisourea, S-methylisothiourea or appropriate substituted isothiourea in alkaline solution.

Still another method for preparing compounds of formula I comprises acylating 6-aminopencillanic acid or an appropriate ester thereof with a reactive functional derivative of an acid of formula IV according to the above described procedures wherein X, R,R and Z are as defined above. This method is not generally favored because the reactants are less readily available than are those of formula III. They can, of course, be prepared by methods known to those skilled in the art.

Examples illustrating the preparation of compounds within the scope of this invention are given below. In the formulae accompanying the examples, APA

represents the moiety S 3 NH CH CH C I l I 0=cNcH- c -o- The guanidino moiety of the acyl side chain, for convenience, employs the following numbering system Tautomeric forms of the guanidino moiety wherein at least one of R R R and R is other than hydrogen are also embraced within this invention.

TABLE I. in Vitro Spectra of Guanidino-Substituted Acyl Derivatives of a-Aminobenzylpenicillin Against Certain Gram-negative Organisms (MIC's) Serr- Proteus atia vulrett-miramiramar- E.co1i Pseudo.aeruginosa garis geri bilis bilis cesens Z 266 104 173 490 A059 8006 C015 C020 A001 K CH NH-C (NH)NH 1 .56 1.56 1.56 1 .56 1 .56 12 .5 1.56 l .56 6. 25 H CH N (CH -C (NI-[)NH 6.25 50 12.5 3. 12 6.25 100 6. 25 6.25 3. 12 H CH CH NH-C (Nl-UNH 3. 12 6. 25 3. 12 3. 12 3. 12 50 0. 78 12 .5 6.25 H

N CH NH 1 12.5 6. 25 3. 12 3. 12 3. 12 100 3. 12 6.25 25 H NH NH-C (NH)NH 3. 12 0. 78 0.78 0.78 6.25 25 3. 12 12.5 3. 12 H .Q-NH-C (NH)NH 3. 12 1. 56 v 1.56 0. 78 3. 12 12.5 6.25 25 12.5 H @-NH-C(NH)NH 6.25 3.12 1.56 6.25 so 50 6.25 25 12.5 H CH NH-C(NH)NH 6. 25 6. 25 3.12 1.56 12.5 l2 .5 25 H 2 l-C(NH]NH 6.25 12 .5 6.25 1.56 6.25 3. 12 6.25 H

CH -NH-C (NHJNHCH 1 56 1. 56 3. 12 0. 78 3. l2 1 56 3. 12 H l CH -N-C (NH) NHCH CH 6. 25 12 .5 12. 3.12 6. 3. 12 12.5 H -NH-C(NH)NH 3. 1 2 5 1.5 l .5 1 .56 0.78 6.25 H

CH NH-C (NHJNH 25 0. 78 POM CH N(CH )-C (Ni-[)NH 12.5 3. l2 12.5 POM CH CH NHC [Nl-UNH 6.25 6.25 0.39 POM Ampicillin 3. 1 200 200 0. 78 6 1 .5 200 H POM hydrochloride salt of pivaloyloxymethyl ester.

The novel penicillins described herein exhibit in vitro activity against a wide variety of both gram-positive and gram-negative bacteria, including indole-positive Proteus. Their useful activity can readily be demonstrated by in vitro tests against various organisms in a brain-heart infusion medium by the usual two-fold serial dilution technique. The in vitro activity of the herein-described compounds renders them useful for topical application in the form of ointments, creams and the like, or for sterilization purposes, e.g., sick room utensils.

The in vitro spectra of a number of 6-[a-(cuguanidinoalkanoyl-amido)acylamido1penicillanic acids of this invention against certain gramnegative bacteria are presented in Table 1 below. D-aaminobenzylpenicillin and 6-[D-2-phenyl-2-(3- Additionally, the compounds of this invention are active versus gram-positive and gram-negative bacteria in vivo via the parenteral route of administration in animals, including man. Their in vivo activity in animals, including man, by the oral route of administration is more limited as regards susceptible organisms. Nevertheless, oral in vivo activity against Escherichia coli and Staphylococcus aureus is a common property of many compounds of this invention. Table ll below presents the comparative in vivo spectra of several compounds within the scope of this invention against D-aaminobenzylpenicillin (Ampicillin). Table II! presents comparative PD values of 6-[D-2-phcnyl-2- (guanidinoacetamido)acetamido1pencillanic acid [A]. 6-[D-2-phenyl-2-(3- guanylureido)acetamido1penicillanic acid [B] and 9 l D-a-aminobenzylpenicillin versus E. 0011', S. aureus and The acute systemic infections in mice were produced Pseudomonas aeruginosa. by the intraperitoneal inoculation of standardized cul- The compounds of Table I] have the formula: tures suspended in hog gastric mucin. Treatment ca c0 APA R l NH I 0 C Z TABLE II. BrVivo Spectrum of Guanidino-Substituted Acyl Derivatives of oiAminobenzylpenicillin Versus Escherichia coli 266. 0 Protection at mg./kg. in MiceVia Subcutaneous Route R=H R=POM* "s Z Protection Protection CH NH-C [NH] Ni-I 100 90 CH N( CH )-C(NH)NH CH CH NH-C (NH) NH 70 CH2 NH-C (NHJNH2 20 CH NH-C (Ni-l) -NHCH 100 CH -NC (NH) NHCH CH 100 Ampici 11in 100 *POM hydrochloride salt of pivaloyloxymethyl ester.

TABLE III Comparative PD Values (m ./kg.) of Compounds A, B and D-aAminobenzyipenicil in Versus Acute Systemic infections in Mice The dual in vivo activity of 6-[D-2-phenyl-2- 0 with the drugs was initiated 0.5 hours after inoculation (guanidinoacetamido)-acetamido]penicillanic acid of the infecting organism. A second dose was adminis- [A] is in surprising contrast to the in vivo activity of the tered 4 hours later. The PD values were calculated structurally related prior art compound 6-[D-2-phenylafter a hold period of 4 days.

2-(3-guanylureido)acetamidolpenicillanic acid [B] Many of the compounds of this invention possess inwhich lacks oral activity against E. coli and S. aureus. ,5 teresting and advantageous pharmacokinetic proper- Further, compounds A and B are active against Pseudoties not found in structurally related penicillanic acid monas aeruginosa whereas D-a-aminobenzylpenicillin derivatives. For example, 6-[D-2-phenyl-2- is inactive. (guanidinoacetamido)-acetamido]pencillanic acid is 1 1 12 shown by parenteral pharmockinetic studies in dogs to compositions may include liquid diluents, for example, be clearly superiot to the structurally related compropylene glycol, diethyl carbonate, glycerol, sorbitol. pound of 6-[D-2-phenyl-2-(3- etc.; buffering agents, as well as local anesthetics and guanylureido)acetamido]penicillanic acid in terms of inorganic salts to afford desirable pharmacological blood levels of drug and in total urinary recover of drug properties.

(Tables IV and V). Many of the penicillin ester compounds of this inven- TABLE W tion exhibit improved absorption on oral administration over that produced by the corresponding free acid or alkali metal salt forms. They, therefore, represent Comparative Average Serum Levels of 6-[D-2-Phenyl-2-(Guanidino-acetamido)AcetamidolPenicillanic [A] Convemem and effectlve dosage forms of the novel and 6-[D-2-Phenyl-2-(3-Guanylureido)Acetamido1Pencillanic Acid i im f f l b [B] in Dogs for a Single Instramuscular Dose of 25 mg./kg. F

urther, many of the esters, especially the acyloxy(- No. of Serum Levels (meg/ml.) at Indicated Hours lower alkyl) esters described herein, although inactive 3 0 l 2 3 4 6 or of relatively low activity against gram-negative or- Pound ganisms per se are, when administered orally to ani- A 4 0 30,4 26,2 10,3 2,1 0 0 mals, including man, metabolized to the parent acid B 4 0 0 0 0 which has a wide spectrum of activity against grampositive and gram-negative bacteria. They thus serve as sources of the parent compounds since they are biolog- Serum samples from a dog administered a single inically converted in vivo to said compounds. The rate of tramuscular dose of 50 mcg./kg. of [A] gave serum metabolic conversion of such esters to the parent acid concentrations of 78 and 48 meg/ml. at one and two occurs at such arate as to provide an effective and prohours post administration. longed concentration of the parent acid in the animal TABLE V Comparative Average Urine Levels of 6-[D-Z-Phenyl-Z-(Guanidinoacetamido)Acetamido]Penicillanic Acid [A] and 6-[D-2-Phenyl-2- (3-Guanylureido)AcetamidolPenicillanic Acid [B] in Dogs Given a single Intramuscular Dose of mg./kg.

of Dose Concentrations in Urine (meg/ml.) at Indicated Hours Com- Recovered 0 0.5 l 2 3 4 5 pound A 35.4 0 366 7l0 1016 554 2I9 I B 9.5 O 26.8 165 205 203 I42 102 The oral and parenteral dosage levels for the herein body. In effect, such esters act as depot sources for the described compounds are, in general, on the order of parent acid. Especially useful in this respect are the from about 25 to 200 mg./kg. of body weight per day acyloxy(lower alkyl) esters such as the benzoyloxand from about 10 to 100 mg./kg. of body weight per ymethyl-, acetoxymethyl-, acetoxyethyl-, pivaloyloxday, respectively. For topical application, the dosage ymethyland a-ethylbutyryloxymethyl esters.

level is on the order of from about 10 to 200 Compounds of formula I in which X is X wherein X mg./kg./day. is, or contains, a substituted phenylene group wherein When used for the purposes described herein, the the substituent is at least one of lower alkyl, lower alkvaluable products of this invention can be used alone 45 oxy, chloro, bromo, fluoro, trifluoromethyl or di(lower or in admixture with other antibiotics or in combinaalkyl)-amino; and those wherein X represents pyrrolyl; tion with apharmaceutica'l carrier selected on the basis as well as those compounds of formula I wherein at of the chosen route of administration and standard least one of R R R or R is alkenyl of from 2 to 6 pharmaceutical practice. For example, they may be adcarbon atoms are also effective antibacterial agents in ministered orally in the form of tablets containing such the same manner as are the products embraced by forexcipients as starch, milk sugar, certain types of clay, mula I above. Such products are prepared by the methetc., or in capsules alone or in admixture with the same ods described herein using as acylating agents the apor equivalent excipients. They may also be adminispropriate a-amino substituted acylamidopenicillanic tered orally in the form of elixirs or oral suspensions acid offormula II and the appropriate guanidino substiwhich may contain flavoring or coloring agents, or be tuted acid derivative of formula III wherein the X and injected parenterally, that is, for example, intramuscu- 3 R6 Variables are as disclosed above larly or subcutaneously. For parenteral administration Additionally, analogous derivatives of 7-aminocephthey are best used in the form of a sterile solution or alosporanic acid, desacetoxy 7-aminocephalosporanic suspension which may be either aqueous such as water, acid, desacetyl 7-aminocephalosporanic acid and terisotonic saline, isotonic dextrose, Ringers solution, or tiary amine derivatives ol7-aminocephalosporanic acid non-aqueous such as fatty oils of vegetable origin (cotwherein the 3-acetoxy group is displaced by a tertiary ton seed. peanut oil, corn, sesame) and other nonamine function as antibacterial agents against both aqueous vehicles which will not interfere with the thergram-positive and gram-negative bacteria. Such derivaapeutic efficiency of the preparation and are non-toxic tives are used in substantially the same manner as are in the volume or proportion used (glycerol, propylene the 6-[a-(w-guanidinoalkanoylg y sorbilOU- Additionally, composiliions suitable amido)acylamidolpenicillanic acid derivatives dcfor extemporaneous preparation of solutions prior to scribed herein. They are prepared by acylation of the administration may advantageously be made. Such appropriate 7-[(a-amino-substitutcd)acylamido]- methods described herein R -(|:HCONH-CH-(EH CH l NH c N c ca In formula V, R, is as defined above and A is selected from the group consisting of hydrogen, hydroxy, acetoxy and teritary amino. Representative of the tertiary amino groups which displace the acetoxy moiety are pyridine, imidazole, benzimidazole, pyrimidine and substituted derivatives of these amines and tri(alkyl)amines (especially those wherein the alkyl group contains from one to six carbon atoms). Additionally, other amines and other nucleophiles such as sulfur compounds (e.g., thiourea, xanthates, dithiocarbamates, mercaptoimidazole, alkyl and aryl mercaptans), and carbon nucleophiles (e.g., indole, N-methyl-indole, resorcinol) also displace the 3-acetoxy group to afford compounds of formula V wherein A represents the nucleophilic agent to afford effective broad-spectrum antibacterial agents. Compounds of formula V, or methods for their preparation, are described in the literature; US. Pat. Nos. 3,560,489 and 3,575,969; French Pat. No. 2,032,408; J. Antibiot. Ser. A 19 (6), 243-9 1966); Cocker et al., J. Chem. 500., 5015-5031 (1969).

Products of formula V can also, of course, be prepared by acylation of 7-aminocephalosporanic acid or related derivatives thereof (desacetoxy, desacetyl, tertiary amine betaine) with an acid of formula IV. However, availability of starting materials renders the above described methods the preferred routes.

The 6-[a-(w-guanidinoalkanoylamido)acylamido]- penicillanic acids of formula I and the analogous derivatives of 7-aminocephalosporanic acid, its desacetyl-. desacetoxyand tertiary amine derivatives, wherein R and R when taken together with the guanidine moiety to which they are attached form an imidazolo group,'

and those wherein one or more of the nitrogen atoms of the guanidino moiety is substituted with lower alkyl, phenyl or benzyl, are also active antibacterial agents. Such compounds, useful for the same purposes and in the same manner as compounds of formula 1, are prepared from appropriate reactants by methods described herein.

The necessary reactants,

. 6 tration of the reaction mixture followed by trlturation ofthe residue with acetone. The product is then hydrolyzed with hydrochloric acid.

. I EXAMPLE I 6-[D-2-Phenyl-2- (Guanidinoacetamido)Acetamido]Penicillanic Acid ca co 5% H l 1 ll 10 0 c CH2 NH c NH2 To a stirred suspension of the triethylamine salt of D-a-aminobenzylpenicillin (1.35 g., 3 mM) in dry N,N-dimethylformamide 12 ml.) at room temperature under a nitrogen atmosphere was added triethylamine (0.42 ml., 3 mM). The mixture was cooled to 0 C., guanidinoacetyl chloride hydrochloride (0.510 g., 3 mM) added and stirring continued for 30 minutes. The cooling bath was removed and the temperature allowed to reach room temperature. Thin layer chromatography in sodium acetate (7 ml. of 0.2M)-acetone (30 ml.) showed an estimated 40% of unreacted D-aaminobenzylpenicillin still present. The mixture was cooled to 0 C. and triethylamine (0.21 ml., 1.5 mM) and guanidinoacetyl chloride hydrochloride (0.255 g., 1.5 mM) added. Stirring and cooling at 0 C. continued for 15 minutes, followed by 30 minutes at room temperature.

The reaction mixture was filtered and the filtrate poured into diethyl ether (300 ml.). The light pink solid which precipitated is collected by filtration and dried in vacuo (2.3 g.). The crude product is suspended in methylene chloride (100 ml.), triethylamine (2.5 ml.) added and the mixture stirred for one-half hour at room temperature, and then filtered to give the title product (0.987 g.; 67% yield).

Guanidinoacetyl Chloride Hydrochloride The hydrochloride salt of guanidinoacetyl chloride was prepared by stirring a suspension of guanidinoacetic acid hydrochloride (1.53 g., 0.01M), chloride hydrochloride methylene chloride ml.) and phosphorous pentachloride (2.08 g., 0.01M) at room temperature under a nitrogen atmosphere overnight. The product, a white solid, was collected by filtration under nitrogen (0.893 g., 47.2% yield).

50 EXAMPLE u 6-[D-2-Phenyl-2- (Guanidinoacetamide)AcetamidolPenicillanic Acid, Pivaloyloxymethyl Ester, Hydrochloride Q Ica c0 5 CH o c0 C(CH Triethylamine (0.42 ml., 3 mM) was added to a solution of D-a-aminobenzylpenicillin, pivaloyloxymethyl ester, hydrochloride (750 mg., 1.5 mM) in dry methylene chloride (12 ml.) under nitrogen and the mixture hydrochloride (202 mg., 1.8 mM) was added and the mixture stirred at C. for minutes, followed by 30 minutes at room temperature. The reaction mixture was again cooled to 0 C., guanidinoacetyl chloride hydrochloride (202 mg., 1.8 mM) added and the mixture then stirred at room temperature for one hour. It was again cooled to 0 C., guanidinoacetyl chloride hydrochloride (85 mg., 0.48 mM) added, the cooling bath removed, and the temperature allowed to rise to room temperature. After stirring for one-half hour, it was evaporated in vacuo and the residue (1.65 g.) triturated with ethyl acetate (100 ml.). The solid was filtered off, the filtrate washed with saturated sodium chloride solution, dried and evaporated in vacuo to give the product (490 mg., 55% yield).

Repetition of this procedure but replacing D-aaminobenzylpenicillin, pivaloyloxymethyl ester, hydrochloride with equimolar amounts of the appropriate acyloxyalkyl ester produces compounds of the formula EXAMPLE lll 6-[D-2-Phenyl-2-(2-Guanidinopropionamide)Acetamido]Penicillanic Acid, Pivaloyloxymethyl Ester, Hydrochloride Triethylamine (0.42 ml., 3 mM) was added to a solution of D-a-aminobenzylpenicillin, pivaloyloxymethyl ester, hydrochloride (1.5 g., 3mM) in dry N,N-dimethylformamide (9 ml.) at room temperature followed by dicyclohexylcarbodiimide (618 mg., 3 mM) and N- amidinoalanine hydrochloride (500 mg., 3mM). The thick reaction mixture was stirred for 1 hour at room temperature after which the dicyclohexylcarbodiimide and N-amidinoalanine additions were repeated and stirring continued for an additional hour. The reaction mixture was filtered and the filtrate poured into diethyl 1 ether (1 liter). The product precipitated and was collected by filtration and air dried. Yield 2.4 g. of light yellow powder.

It was purified by dissolution in ethyl acetate and thorough washing of the solution with saturated aqueous sodium chloride. After drying with anhydrous sodium sulfate the ethyl acetate was removed under reduced pressure to give l.5 g. of product.

N-Amidinoalanine Hydrochloride N-amidinoalanine (3g.) was suspended in diethyl ether (50 ml.) and excess hydrogen chloride gas bubbled in over a 15-minute period. The product was collected as a white gun by decantation of the diethyl ether.

EXAMPLE lV 6[D-2-Phenyl-2-(Guanidinopropionamido)AcetamidolPenicillanic Acid A mixture of D-a-aminobenzylpenicillin, triethylamine salt, (1.35 g., 3 mM) and triethylamine (0.42 ml., 3 mM) in dry N,N-dimethylformamide (17 ml.) was stirred under a nitrogen atmosphere and cooled to 0 C. Guanidinopropionyl chloride hydrochloride (745 mg., 3 mM) was added and the reaction mixture stirred for one-half hour at 0 C., followed by 1 hour at room temperature. The mixture was cooled to 0 C. and triethylamine (0.14 ml., 1 mM) and guanidinopropionyl chloride hydrochloride (186 mg., 1 mM) added. Stirring at 0 C. was continued for one-half hour followed by 1 hour at room temperature. The mixture was then filtered and the clear filtrate poured into diethyl ether (300 ml.). The light pink precipitate which formed was collected by filtration and dried in vacuo (1.6 g.). The dried solid was suspended in methylene chloride (125 ml.), triethylamine (2 ml.) added and the mixture stirred for 1 hour at room temperature. The light pink product was filtered off, washed with methylene chloride and dried in vacuo (1.1 g., yield).

Guanidinopropionyl Chloride Hydrochloride EXAMPLE v 6-[D-2-Phenyl-2-(Guanidinopropionamido)Acetamido]Penicillanic Acid, Pivaloyloxymethyl Ester, Hydrochloride 2 HCl A solution of D-a-aminobenzylpenicillin, pivaloyloxymethyl ester, hydrochloride (1.5 g., 3 mM) in dry methylene chloride (45 ml.) was treated with triethylamine (0.42 ml., 3 mM), stirred and cooled to C. Guanidinopropionyl chloride hydrochloride (560 mg., 3 mM) was added and the reaction mixture stirred at 0 C. for 1 hour. Triethylamine (0.14 ml., 1 mM) and guanidinopropionyl chloride hydrochloride (186 mg., 1 mM) were added and the mixture stirred at 0 C. for 1 hour. The addition of l millimole of each of triethylamine and acid chloride was repeated, followed by an additional hour of stirring at 0 C. One-half millimole of each of triethylamine (0.07 ml.) and acid chloride (93 mg.) were then added, and stirring continued at 0 C. for 1 hour. The reaction mixture was filtered and the filtrate evaporated under reduced pressure to give a white gummy residue. The residue was triturated with ethyl acetate (200 ml.), filtered and the filtrate washed with saturated aqueous sodium chloride (3 X ml.), dried (Na SO and evaporated under reduced pressure to give the title product as a white powder (1.1 g., 63.5% yield).

EXAMPLE VI 6-[D-2-Phenyl-2-(4- Guanidinobutyramido)Acetamido]Penicillanic Acid, Pivaloyloxymethyl Ester, Hydrochloride I l I 0=C- (CH -NHC-NH HCl Triethylamine (0.28 ml., 2 mM) was added to a solution of the hydrochloride salt of D-aaminobenzylpenicillin, pivaloyloxymethyl ester (1.0 g., 2 mM) in dry N,N-dimethylformamide (6 ml.) under a nitrogen atmosphere at room temperature. Dicyclohexylcarbodiimide (412 mg., 2 mM) was then added, the mixture stirred for 3 minutes, and 4-guanidinobutyric acid hydrochloride (362 mg., 2 mM) added. The thick reaction mixture was stirred at room temperature for 2.5 hours, at the end of which one equivalent of each of dicyclohexylcarbodiimide (412 mg., 2 mM) and 4- guanidinobutyric acid hydrochloride (362 mg., 2 mM) was added. Stirring was continued for an additional 4 hours after which the mixture was filtered and the filtrate poured into diethyl ether (300 ml.). The ether was decanted from the pale yellow solid which was then stirred for 1 more hour in a fresh volume of diethyl ether (300 ml.). The solid was collected and dried (1.16 g.).

The dried solid was stirred in methylene chloride (50 ml.) at room temperature for one-half hour, the suspension filtered and the filtrate evaporated to give 1.1 g. of product (88%).

EXAMPLE Vll 6{ l )-2-Phenyl-2-[( 1 -Methylguanidino)Acetamido]Acetamido}-Penicillanic Acid NH p1 NH I To a mixture of D-a-aminobenzylpenicillin, triethylamine salt, (1.35 g., 3 mM) and triethylamine (0.42 ml., 3 mM) in' dry N,N'dimethylformamide (17 ml.) at 0 C. under an atmosphere of nitrogen was added (1 -methylguanidino)acetyl, chloride hydrochloride (600 mg., 3.2 mM) and the reaction mixture stirred at 0 C.

for 15 minutes, followed by 1.5 hours at room temperature. The mixture was re cooled to 0 C., an additional quantity of l-methylguanidino)acetyl chloride hydrochloride (300 mg., 1.6 mM) added and stirring continued at 0 C. for 15 minutes followed by 1.5 hours at room temperature. The reaction mixture was again cooled to 0 C. and 1.6 mM of each-of triethylamine (0.22 ml.) and (1-methylguanidino)acetyl chloride hydrochloride (300 mg.) was added. Stirring was continued at 0 C. for 15 minutes, followed by 2 hours at room temperature. The mixture was filtered and the filtrate slowly poured into diethyl ether (300 ml.) to precipitate a pink powder (1.6 g.). The powder was collected by filtration, dried and triturated in methylene chloride ml.). Triethylamine (2 ml.) was added and the slurry stirred at room temperature for 1 hour and filtered. The filter cake is washed with methylene chloride and dried. Yield 830 mg. (60%.).

(l-Methylguanidino)Acetyl Chloride Hydrochloride Creatine (5 g.) was suspended in diethyl ether (150 ml.) and hydrogen chloride gas bubbled in for 1 hour. The temperature was held at room temperature by external cooling. \The precipitate was filtered off and air dried.

Creatine hydrochloride 1.85 g., 0.01 mole) in methylene chloride (50 ml.) was treated with phosphorous pentachloride (2.06 g., 0.01 mole) at room temperature. The mixture was stirred overnight at room temperature and the acid chloride product filtered off, washed with methylene chloride and dried.

EXAM PLE Vlll 6-{D-2-Phenyl-2-[(1- Methylguanidino)Acetamido1Acetamido}-Penicillanic Acid, Pivaloyloxymethyl Ester, Hydrochloride NH CH NH To a stirred solution of D-a-aminobenzylpenicillin, pivaloyloxy-methyl ester, hydrochloride (1.5 g., 3mM) in dry methylene chloride (50 ml.) was added triethylamine (0.42 ml., 3 mM). The mixture was cooled to 0 C., (l-methy1guanidino)acetyl chloride hydrochloride (560 mg., 3 mM) added and stirring continued at 0 C. for 15 minutes, followed by one hour at room temperature. The reaction mixture was cooled to 0 C. and 3 millimoles of each of triethylamine and acid chloride added. The mixture was stirred at room temperature for 1 hour, re-cooled to 0 C. and acid chloride (280 mg., 1.5 mM) added. It was stirred for 1 hour at room temperature, again cooled to 0 C. and additional acid chloride mg., 0.75 mM) added. The mixture was warmed to room temperature and stirred for 45 minutes. It was filtered, the filter cake washed with methylenc chloride and the combined filtrate and wash solutions evaporated under reduced pressure. The residue was trituratcd with ethyl acetate 125 ml.) and the solid filtered off. The filtrate was washed with saturated salt solution (3 X 20 ml.), dried (Na SO and evaporated in vacuo to give 1.16 g. of product (67%).

EXAMPLE 1x 6-{D-2-Phenyl-2-[(Cl-Methylguanidino)Acetamido]Acetamido}-Penicillanic Acid, Pivaloyloxymethyl Ester, Hydrochloride Tn-co-ApA-cu -o-co-c CH 3 The procedure of Example VI was repeated but using (3-methylguanidino)acetic acid hydrochloride in place of 4-guanidinobutyric acid hydrochloride.

Thus, from 3 millimoles of pivaloyloxymethyl ester of D-a-aminobenzylpenicillin hydrochloride, 3 millimoles of triethylamine and 6 millimoles of each of dicyclohexyl carbodiimide and (3-methylguanidino)acetic acid hydrochloride in 40 ml. of dry N,N-dimethylformamide, an almost quantitative yield of product was obtained.

2-Methylguanidino Acetic Acid N,S-dimethylisothiourea hydroiodide (9.5 g., 0.041 mole)was added portionwise to a stirred solution of glycine (4.45 g., 0.0595 mole) in concentrated ammonium hydroxide (40 ml.) at room temperature. The reaction was stirred overnight and the product recovered by filtration as white crystals: 3.0 g., m.p. 230 C. (dec.).

Repetition of this procedure but replacing the (2- methylguanidino)-acetic acid hydrochloride was therein with an equimolar amount of:

(3-phenylguanidino)acetic acid (3-n-buty1guanidino)acetic acid (l-ethylguanidino)acetic acid 3-guanidinobutyric acid 2-guanidinobutyric acid 3-guanidinopropionic acid (2,3-dimethylguanidino)acetic acid 2-guanidinopropionic acid S-guanidinovaleric acid 3-guanidino-2-methylpropionic acid 2-(B-carboxyethylamino)-2-imidazoline 2-[(carboxymethyl)methylaminol-2-imidazoline 2-(a-carboxypropylamino)-2-imidazoline 2-(carboxymethylamino)-2-(1,4,5,6,-

tetrahydropyrimidine) 2-(e-carboxypentylamino)-2-imidazoline affords the following penicillanic acid pivaloyloxymethyl ester hydrochlorides. For convenience, only the 6-position substituents are listed:

6-{D-2-phenyl-2-[(3- phenylguanidino)acetamido]acetamido} 6-{D-2-pheny1-2-[(3-nbutylguanidino)acetamido]acetamido} 6-{D-2-phenyl-2-[(1- ethylguanidino)acetamido1acetamido} 6-[D-2-phenyl-2-(3- guanidinobutyramido)acetamido] 6-[D-2-phcnyl-2-(2- guanidinobutyramido)acetamido] 6-{D-2-phenyl-2-[2-(2- imidazolinylamino )butyramidolacetamido} 6-{D-2-phenyl-2-[1,4,5,6-tetrahydro-2- pyrimidinyl )aminoacetamido -acetamido} 6-{D-2-phenyl-2-[6-(2- imidazolinyl )aminohexanoylamido acetamido} EXAM PLE X 6-[D-2-Phenyl-2-( 2- lmidazolinylaminoacetam ido)Acetamido Penicillanic Acid NH N I 0=c-cu The procedure of Example V11 was repeated but using N-(2-imidazolinyl)glycyl chloride hydrochloride in place of( l-methylguanidino)acety1 chloride as reactant.

Thus, D-a-aminobenzylpenicillin triethylamine salt (1.35 g., 3 mM), triethylamine (0.77 ml., 5.5 mM) and N-(2-imidazolinyl)glycyl chloride hydrochloride (1.08 g., 5.5 mM) in dry N,N-dimethylformamide (15 ml.) gave 915 mg. (64.5%) of product.

The acid chloride and triethylamine were added in three portions as in Example Vll but at the rate of 3.0, 1.5 and 1.0 millimoles per addition.

N-( 2-lmidazolinyl )Glycyl Chloride Hydrochloride This reactant was prepared from N-(2- imidazolinyl)glycine hydrochloride according to the procedure in Example VII for preparing (1- methylguanidino)acetyl chloride hydrochloride.

5 Repetition of this procedure but using the appropriate a-aminoacylpenicillin and the appropriate cyclic guanidin'o acid of the formula NR u 4 HOOC-X-NH-C-NHR 4-Guanidinobenzoyl chloride hydrochloride (585 mg., 2.5 mM) was added to a stirred mixture of D-aaminobenzylpenicillin trihydrate (1.15 g.. 2.85 mM) and triethylamine (0.35 ml, 2.5 mM) in dry. N,N-dimethylformamide (10 ml.) at 0 C. and under an lm limidarolyl 2-1m 2-( I-imirluzoiinyll THP 2-( l.4.5.(1-lutmhydropyrimidinyll PYR l pyrimidinyl For those compounds wherein R is an ester group, the work-up of procedure V is used.

EXAMPLE Xl 6-[D-2-Phenyl-2-(4-Guanidinoben- 2amido)Acetamido1Penicillanic Acid CIIH-CO-APA-H NH NH 1 n 0- --NH-C-NH2 4-Guanidinobenzoyl Chloride Hydrochloride A suspension of 4-guanidinobenzoic acid (1 g.) in water (25 ml.) was adjusted to pH 1.0 with 6N hydrochloric acid and then warmed to 50 C. The clear solution which resulted was cooled and the white crystalline hydrochloride salt of 4-guanidinobenzoic acid filtered off, washed thoroughly with diethyl ether and dried: 500 mg.; mp. 270 C.

A mixture of the hydrochloride salt thus prepared (1.2 g.) and thionyl chloride (50 ml.) was refluxed for 10.5 hours (care being taken to exclude moisture) and then allowed to stand overnight at room temperature. The title product was recovered as a white powder by evaporation of the excess thionyl chloride.

In accordance with the above acylation procedure, the 4-guanidinobenzoyl chloride hydrochloride is replaced by an equimolar amount of the acid chloride hydrochlorides of the following acids:

2-phenyl-4-guanidinobenzoic acid 2-phenyl-4-guanidinomethylbenzoic acid 4-guanidinomethylphenylacetic acid a-guanidino-octanoic acid a-guanidino-B-phenylpropionic acid 1-guanyl-4-piperidinocarboxylic acid to give the penicillanic acids listed below. Only the 6- position substitucnts are given for convenience:

6-[D-2-phenyl-2-(2-phenyl-4- guanidinobenzamido)acetamido] 6-[D-2-phenyl-2-(2-phenyl-4- guanidinomethylbenzamido)acetamido] 6-[D-2-phenyl-2-(4- guanidinomethylphenylacetamido)acetamido] 6-[D-2-phenyl-2-(a-guanidinooctanoylamido)acetamido] 6-[D-2-phenyl-2-(a-guanidino-B- phenylpropionamido)acetamido] 6-[D-2-phenyl-2-( l-guanyl-4- piperidylcarboxamido)acetamido] EXAMPLE XII 6-[D-2-Phenyl-2-( 3-Guanidinobenzamido)Acetamido]Penicillanic Acid @a m s uu- -NH To a stirred mixture of D-a-aminobenzylpenicillin trihydrate (810 mg., 2 mM) and triethylamine (0.56

3-Guanidinobenzoyl Chloride Hydrochloride A mixture of benzoylcyanamid (1.62 g.), mamiriobenzoic acid (1.37 g.) and ethanol (5 ml.) was heated to 100 C., the ethanol being allowed to evaporate off. When most ofthe ethanol had distilled off, additional ethanol ml.) was added and heating continued until about one-third volume remained. The mixture was then cooled, filtered and the solid product washed first with ethanol, then with ether, and dried (2.4 g.).

The 3-(3-benzoylguanidino)benzoic acid (1.4 g.) thus prepared was refluxed in IN sodium hydroxide (16 ml.) for 1 hour. The reaction mixture was cooled. adjusted to pH 6 and the resulting precipitate filtered off. It was washed successively with water, acetone and ether, and dried (0.558 g. of 3-guanidinobenzoic acid).

A mixture of thionyl chloride (5 ml.) and 3 guanidinobenzoic acid was refluxed for 40 minutes and then evaporated to dryness in vacuo to give the acid chloride.

EXAMPLE Xlll 6-D-2-Phenyl-2-(4-Guanidinocyclohexylcarboxamido)Acetamido Penicillanic Acid Hydrochloride Triethylamine (0.38 ml., 2.66 nM) was added to a stirred suspension of D-a-aminobenzylpenicillin trihydrate (0.536 g., 1.33 mM) in dry N,N-diemthylformamide (5 ml.). The mixture was cooled to 0 C. and the acid chloride of 4-guanidinocyclohexylcarboxylic acid hydrochloride (0.319 g., 1.33 mM) added. The mixture was warmed to room temperature over a 3-hour period. Methylene chloride (65 ml.) was added and the resulting yellow precipitate filtered off, washed with diethylether and dried (0.664 g.). The solid was suspended in methylene chloride (20 ml.), triethylamine (1 ml.) added and the mixture stirred for 1 hour. The suspension was filtered and the product dried in vacuo (0.46] g., 53% yield).

Acid Chloride of 4-Guanidinocyclohexylcarboxylic Acid Hydrochloride A mixture of 4-aminocyclohexylcarboxylic acid (5.26 g., 0.037 M), concentrated ammonium hydroxide (24 mls.) and 2-methyl-2-thiopseudourea sulfate (6.7 g., 0.024 M) was stirred at room temperature for 21 hours. The mixture was cooled in an ice bath and the product, 4-guanidinocyclohexylcarboxylic acid, filtered off, triturated with acetone and dried (0.407 g.).

It was converted to the hydrochloride salt by bubbling hydrogen chloride gas through a suspension of the guanidino acid in ether (40 ml.) for 15 minutes. The salt is filtered off and converted to the acid chloride hydrochloride according to the procedure given in Example XI.

EXAMPLE XIV 6-{D-2-Phenyl-2-[(4-Guanidinophenyl )Acetamido]Acetamido}Penicillanic Acid The procedure of Example XII was repeated but using 4-guanidinophenyl acetyl chloride hydrochloride as acylating agent in place of 3-guanidinobenzoyl chloride hydrochloride and an initial temperature of 70 C. rather than 30C. Yield =2.65 g. (78.5%) ofproduct as a white powder from D-a-aminobenzylpenicillin trihydrate (2.62 g., 6.45 mM), triethylamine (1.8 ml., 12.9 mM) and acid chloride (1.6 g., 6.45 mM) in dry N,N-dimethy1formamide (35 ml.).

EXAMPLE XV 6-{D-2-Phenyl-2-[ (4- Guanidinocinnamoylamido]Acetamido}Penicillanic Acid To a solution of D-a-aminobenzylpenicillin trihydrate (0.921 g., 2.28 mM) and triethylamine (0.64 ml., 4.56 mM) in dry N,N-dimethylformamide (23 ml.) at C. was added a solution of 4-guanidinocinnamoyl chloride hydrochloride (0.593 g., 2.28 mM) in dry N,N-dimethylformamide (5 ml.). The mixture was stirred at 0 C. for 5 minutes and the temperature then allowed to rise to room temperature. Stirring was continued for one-half hour after which the mixture was filtered and the filtrate poured into diethyl ether (800 ml.). The precipitate was collected and dried (1.58 g.). It was suspended in methylene chloride (30 ml. triethylamine (0.5 ml.) added and the mixture stirred for 1 hour. The product was collected by filtration of the slurry, washed with methylene chloride and dried. 1.111 g. (90.7% yield).

The 4-guanidinocinnamoyl chloride hydrochloride was prepared from 4 -aminocinnamic acid according to the procedure of Example X11 for preparing 3- guanidinobenzoyl chloride hydrochloride.

EXAMPLE XVI 6-[ D-2-Phcnyl-2-(4-Guanidinocyclohexylcarboxamido)AcetamidoIPcnicillanic Acid To a solution of 4-guanidinocyclohexane carboxylic (810 mg., 2 mM) in water-N,N-dimethylformamide (12 ml. of 1:1) containing sufficient sodium bicarbonate to give a pH of 7.0. The mixture was stirred for 2 hours at room temperature and then filtered to remove dicyclohexylurea. The filtrate was poured into acetone (2 liters) with stirring and the product recovered by filtration and dried. Repetition of this procedure but replacing 4- guanidinocyclohexane carboxylic acid by an equivalent amount of:

Z-guanidinopalmitic acid 2-(4-guanidinophenoxy)butyric acid 4-guanidinophenoxyacetic acid 4-guanidinomethylphenoxyacetic acid 2-(4-guanidinomethylphenoxy)propionic acid 2-(4-guanidinomethylphenoxy)butyric acid 4-( l-methylguanidino)crotonic acid 2-guanidinocyclohexane carboxylic acid 4-guanidinocrotonic acid 4-(3-n-butylguanidino)crotonic acid 3-guanidinocyclohexane carboxylic acid 2-guanidinocyclopentane carboxylic acid 3-guanidinocyclopentane carboxylic acid 3-guanidinocyclobutane carboxylic acid l-amidino-2-pyrrolidine carboxylic acid 2-imino-4-imidazolidine carboxylic acid l-guanidinocyclobutane carboxylic acid 1-guanidino-2-methylcyclobutane carboxylic acid l-guanidinocyclopentane carboxylic acid l-guanidinocyclopropane carboxylic acid l-guanidinocyclohexane carboxylic acid l-guanidino-3-fluorocyclohexane carboxylic acid 1-guanidino-2-propoxycyclohexane carboxylic acid 1-guanidino-3-nitrocyclohexane carboxylic acid l-guanidinocycloheptane carboxylic acid 1-guanidino-2-ethylcycloheptane carboxylic acid l-guanidinocyclo-octane carboxylic acid respectively, afford the corresponding penicillanic acid derivatives.

EXAMPLE XVll Following the procedures of Examples 1, 11, V11, 1X, X1 and XVl, the penicillanic acids listed below are presubstituted acids or acid halides thereof [ZCOX- -N(R )-C(=NR )NR R acid (370 mg., 2 mM) in N,N-dimethylformamide (1O NH R NR ml.) was added dicyclohexylcarbodiimide (454 mg., l H 4 2.2 mM) in N,N-dimethylformamide (5 ml.) followed 0 C X N C NR R by a solution of D-a-aminobenzylpenicillin trihydrate 5 6 Proc R1 Ra a R4 5 li R X EX.

C H CH3 H C2H5 H CH3 H CH2 1 CGH, CH3 H C5H5 H n-C H H CH2 1 cm. H H CH 0 H, H H CH I H H H H H H H CH l H H H H H H CH 0COCH(C H CH 11 H H H CH3 CH3 CH3 H l,4C,,H l l--C -;H H H H H H CH(CH3)OCOC2H5 CH2 ll n-QH, H H H H CH CH 0CO(4CF C H CHCH l1 n-C H H H CH H H H CH 1 H H H H H H H CH I EXAMPLE XVIII aminomethyla-aminoheptyla-aminopentyla-aminoisobutyla-amino-o-chlorobenzyla-amino-m-chlorobenzyla-amino-p-chlorobenzyla-amino-l ,4-cyclohexadienylmethyla-amino-4-hydroxybenzyla-amino-4iodobenzyloz-amino-3 'lluorobcnzyla-amino-p-methoxybenzyla-amino-Zmitrobenzyla-amino-4-t-butylbenzyla-amino 3-methylbenzyla-amino-4-sulfamylbenzyla-aminol -naphthylmethyla-amino-B-phenethyla-amino-2-furylmethyla-amino-2-thienylmethyla-amino-3-thienylmethyla-amino-3-indolylmethyla-amino-y-methylthiopropyla-amino-y-ethylthiopropyla-aminocyclohexylmethyla-aminocyclopentylmethyla-amino-p-trifluorobenzyla-amino-2-acetamidobenzyll-aminocyclooctyll-aminocyclohexyll-aminocyclobutyll-aminocyclopropyll-aminocyclononyl a-amino-3-hydroxybenzyl a-amino-4-diethylaminobenzyl a-amino-3-bromobenzyl a-amino-a-methylbenzyl respectively, the corresponding [guanidinoalkanoylamidoacylamido]penicillanic acids are produced.

EXAMPLE XIX 6-{D-2-Phenyl-2-[ 2-( 2-Amino-4- lmidazolinylcarboxamido)]Acetamido}-Penicillanic Acid CH co AP A H I NH I C T? N NH NI'I

The hydrochloride salt of the acid chloride of 2- amino-2-imidazoline-4-carb0xylic acid (450 mg., 2.5 mM was reacted with D-a-aminobenzylpenicillin trihydrate (1.15 g., 2.85 mM) according to the procedure of Example XI to give the title product.

The acid chloride of 2 amino-2-imidazoline-4- carboxylic acid was prepared from the hydrochloride salt by treatment with thionyl chloride according to the method of Example XI.

Repetition of this procedure but using 2-amino-2- (1,4,5,6-tetrahydropyrimidine)-5-carboxylic acid hydrochloride in place of 2-amino-2-imidazoline-4- carboxylic acid hydrochloride affords 6-{D-2-Phenyl- 2-[2-(2-amino-5- (l ,4,5 ,6-tetrahydropyrimidinylcarb0xamido)]- acetamido}penicillanic acid.

Repetition of this procedure but replacing D-aaminobenzylpenicillin trihydrate by the a-aminoacylpenicillins listed in Example XVIII produces the corresponding penicillins.

EXAMPLE XX 6-[D-2-Phenyl-2-( l-Guanyl-4-Piperidylcarboxamido)- Acetamido1Penicillanic Acid @wzn-co-AE- NH NH The procedure of Example Xll was repeated but using D-a-aminobenzylpenicillin trihydrate (3.22 g., 8 mM) in N,N-dimethylformamide (30 ml.), triethylamine (224 ml., 1.6 mM), and the acid chloride of 1- guanyl-4-piperidinecarboxylic acid hydrochloride (2.26 g., l mM) in N,N-dimethylformamide ml.). The product was obtained as a light pink powder, 3.4 g., (84%).

l-Guunyl-4-Piperidinecarboxylic Acid Chloride 4-Piperidinccarboxylic acid (6.45 g., 0.05 mole) was dissolved in concentrated ammonium hydroxide (40 ml.) and S-methylisothiourea (6.95 g., 0.05 mole) added. The resulting solution was stirred thoroughly D-a-aminobenzylpenicillin, pivaloyloxymcthyl ester a-amino-3-thienylmethylpenicillin. acetoxymcthyl D-a-amino-4-sulfamylbenzylpenicillin, benzoyloxymethyl ester 19 respectively, affords the corresponding penicillanic acid derivatives.

EXAMPLE XXI 6-[D-2-Phenyl-2-( l-GuanyI-Z-Pyrrolidylcarboxand then allowed to stand overnight at room temperaamido)Acetamido]Penicillanic Acid ture. The product, which precipitated, was filtered off, washed with ethanol and dried; 4.41 g. 52.4% yield). CO APA H The acid chloride was prepared by stirring a mixture of equimolar amounts of the N-guanyl substituted acid NH and phosphorous pentachloride in methylene chloride l overnight at room temperature under an atmosphere of 0 C nitrogen. The methylene chloride was decanted from N the oil which was washed twice with methylene chlo- NH ride and then dried in vacuo to a white gum. 2

Repetition of this procedure but replacing D-a I aminobenzylpenicillin trihydrate by an equivalent A suspension of dicyclohexylcarbodiimide (1.57 g., amount of 7.7 mM), N-hydroxysuccinimide (0.875 g., 7.7 mM),

D-a-amino-3-methylbenzylpenicillin l-guanylproline (1.5 g., 7.7 mM) and N,N-dimethylfor- D-a-amino-4-chlorobenzylpenicillin mamide (50 ml.) was stirred for 2 hours at room tem- D-a-amino-l,4-cyclohexadienylmethylpenicillin 3O perature. The triethylamine salt of D-a- D-oz-amino-3-bromobenzylpenicillin aminobenzylpenicillin (3.36 g., 7.7 mM) was added, D-a-amino-4-sulfamylbenzylpenicillin the mixture stirred at room temperature for 2.5 hours, D-a-amino-4-diethylaminobenzylpenicillin and then filtered. The filtrate was added to ether 1000 D-a-amino-4-trifluoromethylbenzylpenicillin ml.), the mixture stirred, and the ether decanted from D-a-amino-2-acetamidobenzylpenicillin the gummy product. Methylene chloride (250 ml.) and aminomethylpenicillin triethylamine (2 ml.) were added to the gummy proda-aminoheptylpenicillin uct, the mixture stirred for one-half hour, and the solid a-aminocyclohexylmethylpenicillin which formed recovered by filtration and dried over a-amino-Z-thienylmethylpenicillin phosphorous pentoxide (1.9 g.). a-amino-3-thienylmethylpenicillin 40 By means of this procedure but using the appropriate a-amino-2-furylmethylpenicillin a-aminoacylpenicillin and the appropriate l-guanyl a-amino-B-(3-indolyl)ethylpenicillin pyrrolidine carboxylic acid, the compounds listed a-amino-B-phenethylpenicillin below wherein R=H are prepared. Those compounds a-amino-y-phenylpropylpenicillin wherein R is an acyloxyalkyl group are prepared by the a-amino-l -naphthylmethylpenicillin procedure of Example VI from the appropriate penicila-amino-a-methylbenzylpenicillin lin acyloxyalkyl ester. a-amino-sec-butylpenicillin R a-amino-ethylthioethylpenicillin 2 a-am no-w-ethylth|opentylpen1c1llm R C CO APA R l-ammo-l -cyclohexylpeniclllln l-amino-l -cyclobutylpenicillin NH NR a-amino-4-hydroxybenzylpenicillin I H 4 a-amino-3-hydroxybenzylpenicillin 0 C N C NR R RI R2 R4 R5 e R lsomcr C,.H,, H H H H H 3 CH5 H H (.H. H (ll .()(()(ll((' H 2 0H5 H H ('H H (H (X()(H((',H.-.)-e 3 CHH; H (,;H,-, H ('-,H,, H 2 C(IHE H CH H H 3 3ClC,;H H H H H H 2 4CF;,C"H4 H (."H ('H; H H 2 2-CH;,CONHCH4 H H H C.,H H 3 2-NO,C.;H4 H H H H CH(CH,-,)()(()(ZH,, 2 4-BrC,;H H H H (3H5 H 3 4HOC5H4 H H H H H 3 4HOC(,-H4 H H CH" H CHQOCOQCH 2 2 5)2 li -t H H CH3 CH3 H 2 1 1 H H H H H 2 C6H5CH2CH2 H H H H H 3 4(H2NSO2)CGH4 H H H H H 3 CH9 H H C-ZH H H 2 lsomcr H z CHHHHCHHHHHHHHHHHHCHHHHH EXAMPLE XXII 6-[D-2-Phenyl-2-(3-Methylguanidin0)Acetamido CH-CO-APA-H Acetamido]Penicillanic Acid CH-CO-APA-H l NH inn 2 NH- 0 NH(CH3) Following the procedure of Example I but using l-guanyl-3-piperidine carboxylic acid hydrochloride as 35 acylating agent in place of a-guanidinophenylacetyl chloride provides 482 mg. (48% yield) ofthe title product.

lsomer HHHHCHHHHHCHHHHHHHH "m HHCHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHHHH The esters listed above are prepared from appropriate reactants by the procedure of Example VI.

EXAMPLE XXIV 6-{D-2-Phenyl-2-[ l-( 2-Iminoimidazolidino)- ]Acetamido}-Penicillanic Acid NH NH l 30 l l To a stirred solution of triethylamme (2.79 ml., 0.02 HOOC X N (CH NH C NH. M) in dry N,N-dimethylformamide (100 ml.) was P added D-a-aminobenzylpenicillin trihydrate (4.03 g., 0.01 M). When solution was complete, the reaction mixture was cooled to 0C. and the acid chloride of 1- 35 1 I CO L R carboxymethyl-2-iminoimidazolidine hydrochloride NH NH (1.97 g., 0.01 M) added. The mixture was stirred at 0 I C. for ten minutes followed by 1.5 hours at room tem- 0 C X N NH per-ature. It was then filtered and the filtrate poured into diethyl ether (2 liters). The solid which precipi- 40 CH tated was filtered off under an atmosphere of nitrogen C 2 R. R x R p 5 CH2 H 3 s s CH2 CH OCOC(CH 2 fi h H -M5 H I q i 3 4-BrC H H H. H 3

4-HOCGH4 H CH2 H 3 4-H( )C6H4 H CHZCH H 2 3-th1enyl H H2 H 2 3-thienyl H l,4CH C ,H., H 2 Z-thienyl H CH H 2 Z-furyl H CH2 CH2OCOCH3 3 4-(CH ,)2NC,;H4 H (CH2)5 H 3 s s H (CH2)5 H 2 ti s H (CH-H1 H 2 G H H H 3 CGHH H l,4C H H 2 CH1 H 1,4'-CBH,Q H 2 c n H I,4CH=CHC H H 2 4-ClC H H l,4C H, H 3 4-(CZH5O)CGH4 H 1,3C H H 3 C6H5 H l.4CH C H H 3 4-C H C H H l,4CH C ,H H 3 8 5 H CH==CHCH2 H 2 l.4-cyclohexadienyl H l,4-CH=CHC H H 2 a 5 H l,4CH OC H CH(CH;,)OCOCH;, 3 ECHZCHQC- CH2 H 3 -((H-:l.\" ('I'K'Il ll 3 ((11,) ('H,('ll-, H 3 H H (1|, (lI(('ll ()((K,H 2 H H 1.4 r n. H 3 CH3 H ('HA'HZ H 3 E25,, 5H3 l.3(. H H 2 CH=C V CZHQaSCmCH 2 H 2 H (H t H ocoou- Continued R R2 X R p 3-indolylmethyl H CH2 H 3 C,H H (CH H 3 s s CH3 CH2 H 2 l.4 cyclohexadienyl H CH2 H 2 l.4-cyclohexadienyl H CH H 3 l-naphthyl H CHCH; H 3 C6H,, (2H5 CH2 H 3 t-H Nso C H H CH CH OCO(- 2 4 c1c..H. 3-lhienyl H 1 4 CH= H 3.

CHC"H4 3-thienyl H l 3-CH= H 3 (H-C..H, 3-thienyl H l 2CH=)\ H 3 CHC.,H 3--N()2C,;H4 H l 2CH= H 2 CH-QH 4 HOC,;H;\ H l,3-CH= H 2 CHC6H4 CGH H l ,4CH2OCBH4 H 2 The above listed estersare prepared from the appro- Continued pr1ate acyloxyalkyl pen1c1ll1n esters by the procedure of R1 R: R 11 Example V].

3-thier1yl H CH2OCOCH(C2H5)2 0 EXAMPLE XXV 2-furyl H CHQOCOCZHS l 3-indolylmethyl H CH(CH -,)OCOC2H5 l 6-{D-2-Phenyl-2-[2-(2-Am1no-4- H scH cH S CH OCOCH 0 C H CH OCOC(CH) 0 lmlda zol1nylcarboxam1do)lficetami ldoi} Pimc lljamc 6 (CH2)? CHiOCOCmCiIZh 0 Acid, Plvaloyloxymethy ster, y roc on e (CH2)? CH OCOCH 0 Z-Amino-2-imidazoline-4-carboxylic acid hydrochloride (3 mM) is reacted with D-a- 3O aminobenzylpenicillln, pivaloyloxymethyl ester, hydrochloride according to the procedure of Example Ill to produce the title compound.

In like manner, the following compounds are prepared from appropriate acyloxyalkylpenicillin esters EXAMPLE XXVI and 2-amino-2-imidazoline-4-carboxylic acid and 2- amino-2-( l ,4,5,o-tetrahydropyrimidine)carboxylic acid hydrochlorides.

6-{D-2-Phenyl-2-[(3-Allylguanidino)Acetamido1Acetamido}Penicillanic Acid To D-a-aminobenzylpenicillin, triethylamine salt (7.02 g., 15.6 mM) and triethylamine (1.09 ml., 7.8 mM) in dry N,N-dimethylformamide (24ml.) at 0 C. was added (3-allylguanidino)acetyl chloride hydrochloride (1.645 g., 7.8 mM). The mixture was stirred (3-Allylguanidino)Acetyl Chloride Hydrochloride A solution of allylthiourea (23.2 g., 0.2 m), methyl iodide (3.3 g., 0.3 M) and absolute methanol ([00 ml.) was refluxed for two hours and then evaporated in vacuo to an amber glass which crystallized when scratched with a glass rod. The solid was pulverized and triturated with diethyl ether (500 ml.) to give S- 

1. A compound of the formula
 1. A COMPOUND OF THE FORMULA
 2. A compound according to claim 1 wherein R1 is phenyl; each of R2, R3, R4, R5, R6 and R is hydrogen; and X is alkylene.
 3. A compound according to claim 1 wherein R1 is phenyl; each of R2, R3, R4, R5 and R6 is hydrogen; R is lower alkanoyloxy lower alkyl; and X is alkylene.
 4. A compound according to claim 1 wherein R1 is phenyl; each of R2, R3, R4, R5, R6 and R is hydrogen; and X is phenylene.
 5. A compound according to claim 1 wherein R1 is phenyl; each of R2, R3, R4, R5, R6 and R is hydrogen; and X is phenylenemethylene.
 6. A compound according to claim 1 wherein R1 is phenyl; R3 is methyl; each of R2, R4, R5, R6 and R is hydrogen; and X is alkylene.
 7. A compound according to claim 1 wherein R1 is phenyl, R3 and R5 when taken together with the guanidino moiety to which they are attached are imidazolidino; X is methylene; and each of R2, R4, R6 and R is hydrogen.
 8. A compound according to claim 1 wherein R1 is 1,4-cyclohexadienyl; each of R2, R3, R4, R6 and R is hydrogen; R5 is methyl; and X is methylene.
 9. The compound according to claim 2 wherein R1 is phenyl, each of R2, R3, R4, R5, R6 and R is hydrogen; and X is methylene.
 10. The compound according to claim 3 wherein R1 is phenyl, each of R2, R3, R4, R5 and R6 is hydrogen; R is pivaloyloxymethyl; and X is methylene.
 11. The compound according to claim 4 wherein R1 is phenyl; each of R2, R3, R4, R5, R6 and R is hydrogen; and X is 1,4-phenylene.
 12. The compound according to claim 5 wherein R1 is phenyl; each of R2, R3, R4, R5, R6 and R is hydrogen; and X is 1,4-phenylenemethylene. 